Triglyceride hydrolysis and assay

ABSTRACT

GLYCEROL ESTERS PRESENT IN AQUEOUS MEDIA, SUCH AS SERUM TRIGLYCERIDE AND MILK FAT, ARE ASSAYED BY COMPLETE ENZYMATIC HYDROLYSIS USING BOTH A LIPASE AND A PROTEASE, WHEREUPON THE LIBERATED GLYCEROL IS DETERMINED. A NUMBER OF ALTERNATIVE ENZYMATIC PROCEDURES ARE PROVIDED FOR THE GLYCEROL ASSAY, ALL IN THE ORGINAL AQUEOUS MEDIUM, SUCH AS CONVERSION TO GLYCEROL-1-PHOSPHATE WITH GLYCEROL KINASE; CONVERSION OF ATP TO ADP BY THE SAME RECTION FOLLOWED BY CONVERSION OF THE ADP PLUS PHOSPHOENOLPYRUVATE USING PYRUVATE KINASE TO ATP AND PYRUVATE ION FOLLOWED BY DETERMINING THE LATTER; AND ALTERNATIVELY CONVERTING THE PYRUVATE ION SO FORMED WITH NADH USING LACTATE DEHYDROGENASE TO LACTATE ION AND NAD AND DETERMINING THE LATTER BY OPTICAL MEASUREMENT.

United States Patent 3,703,591 TRIGLYCERIDE HYDROLYSIS AND ASSAY Giovanni Bucolo, San Diego, and Harold David, Los glgsles, Calif., assignors to Calbiochem, Los Angeles,

1 N0 Drawing. Filed Dec. 16, 1970, Ser. No. 98,904 Int. Cl. ClZk 1/04 US. Cl. 195-103.5 R 35 Claims ABSTRACT OF THE DISCLOSURE Glycerol esters present in aqueous media, such as serum triglyceride and milk fat, are assayed by complete enzymatic hydrolysis using both a lipase and a protease, whereupon the liberated glycerol is determined. A number of alternative enzymatic procedures are provided for the glycerol assay, all in the original aqueous medium, such as conversion to glycerol-l-phosphate with glycerol kinase; conversion of ATP to ADP by the same reaction followed by conversion of the ADP plus phosphoenolpyruvate using pyruvate kinase to ATP and pyruvate ion followed by determining the latter; and alternatively con verting the pyruvate ion so formed with NAD-H using lactate dehydrogenase to lactate ion and NAD and determining the latter by optical measurement.

This invention relates to a rapid method of determining fatty acid glycerol esters in various aqueous media, such as serum, milk, and the like.

In many fields, but particularly in clinical biochemistry, it is important to determine the content of fatty acid esters of glycerol in a particular sample. The assay is of particular importance in human serum, where elevated levels are of great diagnostic value. Methods depending upon determination of the fatty acid moiety, and methods in which glycerol liberated from other sources contributes to' the total assay, have various disadvantages. A rapid, accurate method operating exclusively and at the same time completely on the fatty acid glycerol esters is an important desideratum in the clinical field, and prior art methods are in general not fully satisfactory.

An object of the present invention is to provide a rapid and accurate procedure which liberates glycerol from its esterified form as a fatty acid ester, for example when present in aqueous media such as serum, and which permits the glycerol to be assayed by a variety of alternative methods without the necessity of isolating the glycerol from the medium under test.

Other objects of the invention will appear as the description thereof proceeds.

Generally speaking, and in accordance with illustrative embodiments of our invention, we add to an aqueous medium containing the fatty acid glycerol to be assayed, a mixture of first a lipase, which may be of plant or animal origin, but among which we prefer and find best a microbial lipase, such as the lipase from Chromobacterium viscosum, variant paralipolyticum, crude or purified, the lipase from Rhizopus delemar, purified, for example as noted in Fukumoto et al., J. Gen. Appl. Microbiol., 10, 257-265 (1964); and lipases having similar activity; and second, a protease. Proteases in general maybe used, such as by way of example and not by limitation, chymotrypsin, trypsin, Streptomyces griseus protease (commercially available under the registered trademark Pronase), elastase, papain, and bromelin. Mixtures of these may also be employed. This simple enzyme mixture is in general sufficient, although the hydrolysis may generally be expedited somewhat by the simultaneous inclusion of a simple protein such as serum albumin, egg albumin, globulins, and the like.

As will be set forth in detail hereinbelow, the glycerol liberated by the action of the enzyme mixture just described may be assayed in a number of ways, although we prefer one in particular which will now be described.

We prefer to carry out the enzymatic hydrolysis of the triglycerides by the lipase-protease combination as just described in the presence of the components of three additional enzymatic transformation systems, whereby first, the glycerol which has been liberated is converted to a-glycerol phosphate by glycerol kinase with the simultaneous conversion of adenosine triphosphate to adenosine diphosphate; second, the conversion of the latter back to adenosine triphosphate with the simultaneous conversion of phosphoenolpyruvic acid to pyruvate ion under the action of pyruvate kinase; and third, the conversion of the pyruvate ion to lactate ion with the simultaneous conversion of nicotinamide adenine dinucleotide from its reduced to its oxidized form, under the action of lactate dehydrogenase. All of these system components are present simultaneously in the mixture, so that as the hydrolysis of the triglyceride proceeds, the optical density of the solution at 340 nanometers decreases as a result of the oxidation of the dinucleotide. This sequence of enzymatic conversions is indicated diagrammatically as follows:

enzymatic Trlgiycerldes---- GlyceroH- F. F.A.

hydrolysis LDH Pyruvate+NADH Lactate-l-NAD colored I.e., absorbs strongly at 340 nm.; molar extinction coefficient arr:6.22 X 10 2 I.e., transparent at 340 nm.

Abbreviations A useful and preferred embodiment of our invention comprises an assay mixture containing the preferred constituents to carry out a single triglyceride assay in accordance with the invention. Inasmuch as the preferred procedure involves the enzymatic hydrolysis as already described using the enzyme combination disclOsed, followed by the conversion of the liberated glycerol as likewise described, it is convenient to furnish the assay mixture in two separate containers, such as glass vials, one of which, which may be termed Vial A for convenience, may contain all the components needed for the assay except the glycerol kinase. The latter may be placed in Vial B. Of course, other distributions of the components are possible, any of those involved solely with glycerol conversion and subsequent steps being includable in Vial B if desired. However, we prefer and find best the following reaction mixture:

Vial A Potassium phosphate buffer, 0.1 M, pH 7 Magnesium aspartate: 1.6 mg.

ATP disodium: 0.9 M

Phosphoenol pyruvate: 0.9 M

Bovine serum albumin: 5.0 mg.

NADH to a final absorbance of 0.8 (optical density at LDH: 2 LU.

Pyruvate kinase: 6 I.U.

u-Chymotrypsin: 1100 NLF. units Lipase, Rlzizopus delemar: 1200 lipase units (Total volume: 3 ml.)

(One (1) lipase unit is the quantity of enzyme which will release fatty acids from a substrate of triolein to require 1 ml. of 0.05 N potassium hydroxide for neutrali zation after a 30-min. incubation period at 37 C.)

(One (1) N.F. unit of tar-chymotrypsin is that quantity of enzyme which will produce an absorbance change of 0.0075/minute at 237 nanometers when incubated with a substrate of N-acetyl-L-tyrosine ethyl ester under the conditions of assay.)

Vial B Glycerol 'kinase: 2 I.U. (International Units) The assay in accordance with the invention is carried out by adding an aliquot of liquid containing the triglyceride to be assayed, which may be for example 50 #1. of serum, to the 3 ml. contents of Vial A. This is then incubated at between 25 C. and 37 C. for approximately ten minutes. The optical density is then determined at 340 nm. Then the 2 I.U. of glycerol kinase contained in Vial B is added, and the mixture allowed to stand for an additional minutes at the same temperature, whereupon the optical density is determined again. The difference is proportional to the triglyceride content of the aliquot.

It may be noted that the essential contents of the reaction mixture in accordance with the invention, especially as packaged for single assays, comprises a microbial lipase; a protease; pyruvate kinase; lactate dehydrogenase; NADH; ATP; Phosphoenol pyruvate; a magnesium ion source; a buffer; and glycerol kinase.

An alternative procedure consists in omitting the NADH and the lactate dehydrogenase from the mixture described above, so that only the first three reaction steps depicted hereinabove occur; and adding a sufiicient quantity of dinitrophenyl hydrazine to react with the pyruvate ion formed in the aforementioned third reaction step. The reaction product is colored when alkalized, and may be readily measured by a colorimeter.

A further alternative procedure is to omit the phosphoenol pyruvate; the pyruvate kinase; and the lactate dehydrogenase from the reaction mixture previously described, and to add instead glycerol phosphate dehydrogenase, whereupon the a-glycerol phosphate and the NAD are converted respectively to dihydroxyacetone phosphate and NADH. The amount of NADH formed, which is proportional to the amount of triglyceride originally present, can conveniently be measured by the increase in fluorescence. An alternative sub-procedure is to include also a sufficient quantity of dinitrophenyl hydrazine in this system as well which will result in the formation of a colored reaction product with the dihydroxyacetone phosphate liberated. The amount of the reaction product can then readily be determined with a colorimeter and is likewise a measure of the triglyceride originally present.

A still further alternative procedure is to utilize a reaction mixture which provides only the enzymatic conversion to glycerol which is the first reaction step set forth in the tabulation hereinabove. The reaction mixture, however, also includes NAD and glycerol dehydrogenase. As a result, the glycerol which is liberated in the first step is converted to dihydroxyacetone with the simultaneous production of NADH. The increase in optical density at 340 nm. then becomes a measure of the quantity of triglyceride originally present, as previously described. A suitable glycerol dehydrogenase is that obtainable from Enterobacter aerogenes; this enzyme is commercially available. An alternative subprocedure here is to add dinitrophenyl hydrazine, which forms a colored reaction product with the dihydroxy acetone, so that the latter may then be measured colorimetrically.

In the two alternative procedures just described, viz, in the first of which glycerol phosphate dehydrogenase and in the second of which glycerol dehydrogenase, respectively, are used, an equivalent amount of NADH is formed, as already mentioned. A further alternative subprocedure applicable to both of these is to utilize the known behavior of variou tetrazolium salts, which upon reduction are converted from colorless, water-soluble compounds to colored dyes. The NADH which is quantitatively formed from the triglyceride may be caused to transfer its hydrogen (becoming oxidized in the process) to the tetrazolium salt, again quantitatively, through the mediation of any of several known substances, among which we prefer especially diaphorase or, alternatively, phenazine methosulfate. The amount of dye thus formed may be readily measured by colorimetry, i.e., by carrying out optical density change measurements in the visible region.

It is not believed necessary to spell out the details of these alternative sub-procedures just described, since they are fully documented in the literature. Representative articles, which together with the literature cited therein are hereby incorporated herein by reference, are the following:

American Journal of Clinical Pathology, vol. 45, No. 5, May, 1966: Rapid Colorimetric (Tetrazolium Salt) Assay for Lactate Dehydrogenase, by R. O. Briere, J. A. Preston, and I. G. Batsakis.

Methods of Enzymatic Analysis, by H. U. Bergmeyer, New York: Academic Press, 1965; pages 953-955.

Coming now to the relative proportions of the selected lipase (or mixture of selected lipases) and the selected protease (or mixture thereof), we prefer that for each 1000 lipase units present in the assay mixture, there be present from about 5 to about 500 international units of protease. We find best a subrange therein of about 20 to about LU. (international units) of protease.

The lipase unit has been defined hereinabove. The international unit of proteolytic activity is the amount of protease which causes a turnover of one micromol per minute of a substrate which is specific for the particular enzyme in question, under conditions approximating an optimum for the system considered. Thus, for cymotrypsin the substrate is tyrosine ethyl ester, and the turnover rate may be determined in any number of ways, as by the change in optical density at 237 nm., or by determining the amino acid liberated, as phenol reagent tyrosine equivalents or by formol titration.

The N.F. (National Formulary) unit is occasionally used for proteases, and appears in an example hereinabove. Since 1 I.U. unit is equivalent to 28 NP. units, it will be seen that the 1100 NR units of chymotrypsin in the example is equal to 39 I.U. Since 1200 lipase units were present in the exemplary mixture, it will be seen that for each 1000 lipase units, our example shows about 32 LU. of protease.

It will be clear from the foregoing that in distributing the components of our preferred assay mixture between Vial A and Vial B, one of the vials, e.g., Vial A, should contain at least the lipase and the protease; whereas the second vial, e.g., Vial B, should contain at least the glycerol kinase. The remaining components may be distributed as desired between the two vials. Our preferred distribution, however, has been set forth hereinabove.

In proceeding in accordance with the invention as has been disclosed hereinabove, it will be found that the glycerol esters are completely hydrolyzed, so that a stoichiometric amount of glycerol is liberated, as indeed has already been explained. We are unable to offer an explanation of the underlying mechanism whereby this is accomplished, but it is clear that it stems from the conjoint presence of the lipase with the protease, all as described and specified. For example, when proceeding in accordance with the detailed example given hereinabove, and when the aqueous medium under test is human serum, it is found that the glycerol liberated is that quantity to be expected on the basis of complete hydrolysis of the triglycerides present, the latter being determined by standard procedures well known in the art.

Those skilled in the art will recognize that a-glycerol phosphate may also be named as glycerol-l-phoshpate; and that the fatty acid glycerol esters in serum may be and generally are referred to simply as triglycerides.

As will be clear from the explanations given hereinabove, one unit of lipase means the amount of lipase equivalent to one lipase unit as specified hereinabove.

We wish it to be understood that we do not desire to be limited to the exact details of components and procedures shown and described, for obvious modifications will occur to a person skilled in the art.

What we claim is:

1. In a process of assaying an aqueous liquid containing a fatty acid glycerol ester for its content of said ester in which said ester is hydrolyzed to liberate all of said glycerol followed by determining the amount of glycerol present, the improvement which consists in effecting said hydrolysis by adding both a lipase and a protease to said liquid, whereby substantially complete hydrolysis of said ester is caused to take place.

2. A process in accordance with claim 1 wherein said lipase is microbial.

3. A process in accordance with claim 2 wherein said lipase is chosen from the class. consisting of Rhizopus delemar and Chromobacferium viscoswm lipases, and mixtures thereof.

4. A process in accordance with claim 3 in which said protease is chosen from the class consisting of chymotrypsin, trypsin, streptomyces griseus protease, elastase, papain, bromelin, and mixtures thereof.

5. A process in accordance with claim 4 wherein from about 5 to about 500 LU. of said protease is present for each 1000 units of said lipase.

6. A process in accordance with claim 2 wherein from about 5 to about 500 LU. of said protease is present for each 1000 units of said lipase.

7. A process in accordance with claim 1 wherein said protease is chosen from the class consisting of chymotrypsin, trypsin, streptomyces griseus protease, elastase, papain, bromelin, and mixtures thereof.

8. A process in accordance with claim 1 wherein from about 5 to about 500 I.U. of said protease is present for each 1000 units of said lipase.

9. A process in accordance with claim 1 wherein said liquid is serum, and said glycerol ester is serum triglyceride.

10. A process in accordance with claim 1 wherein, subsequent to said liberation of glycerol, adenosine triphosphate and glycerol kinase are added to said liquid in sufficient quantity to convert said glycerol to glycerol-l-phosphate and said adenosine triphosphate to adenosine diphosphate; and in which said adenosine diphosphate is assayed, whereby the original content of said fatty acid glycerol ester may be determined..

11. A process in accordance with claim wherein to said aqueous liquid containing said glycerol-l-phosphate, nicotinamide adenine dinucleotide and glycerol phosphate dehydrogenase are added in sufficient quantity to convert said glycerol-l-phosphate and said NAD to dihydroxyacetone phosphate and nicotinamide adenine dinucleotide, reduced (NADH) respectively.

12. The process in accordance with claim 1 wherein said NADH is assayed by fluorimetry.

13. The process in accordance with claim 11 wherein to said aqueous liquid containing said NADH, a tetrazolium salt is added together with a hydrogen transfer agent for said tetrazolium salt, whereby the latter is quantitatively converted to a colored dye with concomitant oxidation of said NADH.

14. The process in accordance with claim 13 wherein said hydrogen transfer agent is selected from the class consisting of diaphorase and phenazine methosulfate.

15. A process in accordance with claim 10 wherein to said aqueous liquid containing said glyceroll-phosphate in said ADP, there are added phosphoenol pyruvate and pyruvate kinase in sufficient quantity to convert said phosphoenol pyruvate to pyruvate ion, and in which said pyruvate ion is assayed to give an indication of the amount of said glycerol ester originally present.

16. A process in accordance with claim 15 wherein said pyruvate is assayed by adding to said aqueous liquid a sufiicient quantity of dinitrophenyl hydrazine to react therewith, whereby a colored compound is formed.

17. A process in accordance with claim 15 wherein to said aqueous liquid containing said pyruvate ion there are added NADH and lactate dehydrogenase in suflicient quantity to convert said pyruvate and said NADH to lactate ion and NAD respectively.

18. The process in accordance with claim 17 in which the concentration of said NAD in said aqueous liquid is determined by measuring the change in optical density thereof at approximately 340 nanometers.

19. The process in accordance with claim 1 wherein, subsequent to said liberation of glycerol, adenosine triphosphate and glycerol kinase are added to said liquid in sufficient quantity to convert said glycerol to glycerol-1- phosphate and said adenosine triphosphate to adenosine diphosphate; and in which said gycerol-l-phosphate is assayed, whereby the original content of said fatty acid glycerol ester may be determined.

20. A process in accordance with claim 19 wherein said glycerol-l-phosphate is assayed by adding to said liquid NAD and glycerol phosphate dehydrogenase, whereby said glycerol-l-phosphate is converted to dihydroxyacetone phosphate and said NAD is converted to NADH; and wherein at least one of said conversion products is assayed to give a measure of the fatty acid glycerol ester originally present.

21. The process in accordance with claim 20 wherein said NADH is measured by determining the increase in fluorescence of said solution.

22. The process in accordance with claim 20 wherein dinitrophenyl hydrazine is added to said liquid, whereby a colored reaction product is formed with said dihydroxyacetone phosphate and wherein the intensity of color produced in said solution is determined as a measure of the dihydroxyacetone phosphate.

23. The process in accordance with claim 1 wherein to said liquid there is added NAD and glycerol dehydrogenase, whereby said glycerol is converted to dihydroxyacetone and said NAD is converted to NADH; and in which said NADH is assayed to give a measure of the fatty acid glycerol ester originally present.

24. The process in accordance with claim 23 wherein to said aqueous liquid containing said NADH, a tetrazolium salt is added together with a hydrogen transfer agent for said tetrazolium salt, whereby the latter is quantitatively converted to a colored dye with concomitant oxidation of said NADH.

25. The process in accordance with claim 24, wherein said hydrogen transfer agent is selected from the class consisting of diaphorase and phenazine methosulfate.

26. The process in accordance with claim 23 wherein said glycerol dehydrogenase is derived from Enterobacteraerogenes.

27. The process in accordance with claim 23 wherein said NADH is assayed by determining the increase in optical density at 340 nm.

28. A reagent combination for the analysis of fatty acid glycerol esters which comprises a first vial containing a lipase and a protease; a second vial containing glycerol kinase; and pyruvate kinase; lactate dehydrogenase; NADH; ATP; phosphoenol pyruvate; a magnesium ion source; and a buffer in any preselected distribution in said vials.

29. A reagent combination in accordance with claim 28 wherein from about 5 to about 500 LU. of said protease is present for each 1000 units of said lipase.

30. A reagent combination in accordance with claim 29 wherein said lipase is a microbial lipase.

31. A reagent combination in accordance with claim 28 wherein said lipase is a microbial lipase.

32. A reagent combination in accordance with claim 28 wherein said lipase is derived from Rhizopus delemar; said protease is a-chymotrypsin; said magnesium ion References Cited UNITED STATES PATENTS 10/1950 Halmbacher 195-3 R OTHER REFERENCES George Guilbault, Enzymatic Methods of Analysis, 1st ed., Pergamon Press, p. 64, 1970.

A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner 

